- Email: [email protected]
Blood-brain transport and regional distribution of bromo-benzodiazepine
Brain Research, 401 (1987) 55-59 Elsevier
55
BRE 12263
Blood-brain transport and regional distribution of bromo-benzodiazepine Lester R. D r e w e s l, G u n t e r Mies 2, K.-A. H o s s m a n n 2 and G e r h a r d St6cklin 3 1Department of Biochemistry, School of Medicine, University of Minnesota, Duluth, MN55812 (U.S.A.), 2Max-Planck Institute for Brain Research, Cologne and 3Institute of Chemistry, KFA Jiilich, Jiilich (F.R. G.) (Accepted 27 May 1986)
Key words: Benzodiazepine; Transport; Blood-Brain; Receptor; Blood flow; Autoradiography; Integral method
The blood-brain transport and regional distribution of a tritium-labeled, brominated benzodiazepine (BFB) was determined for the rat brain in vivo. The unidirectional transport constant from blood to brain was measured by a graphical, integral method and was found to be 0.83 ml/g/min, a value which indicates that transport is essentially flow-dependent. The apparent volume of distribution increased linearly during the measurement period, suggesting that back transport from brain to blood was zero and that BFB was trapped in the tissue, possibly by specific receptors or acceptors. Under the conditions of these experiments, autoradiography of brain tissue sections indicated a regional distribution of [3H]BFB similar to that expected for regional cerebral blood flow. These results indicate that BFB is a useful blood flow tracer in brain and suggest that BFB radiobrominated with bromine-75 may, under appropriate conditions, be a suitable tracer for in vivo regional blood flow measurement or benzodiazepine receptor mapping by positron-emission tomography.
INTRODUCTION During the past two decades benzodiazepine ( B Z D ) drugs such as d i a z e p a m have gained remarkable popularity as anxiolytics, sedative anticonvulsants, and muscle relaxants. In recent years their mechanisms of action have b e e n investigated, and recent studies have focused on specific high-affinity binding sites for these drugs in the m a m m a l i a n nervous system 11'a5. These binding sites now a p p e a r to be linked in some yet undefined way to the postsynaptic G A B A r e c e p t o r and associated chloride ionophore 17. In vitro binding studies have shown regional differences in the distribution of B Z D binding sites. Cerebral cortex has about 2.5 times the r e c e p t o r density as the cerebellum 2'12. H o w e v e r , few in vivo studies have been r e p o r t e d 9. Recently, 1,4-benzodiazepines were labeled with the isotope [75Br] as potential agents for mapping receptors in vivo 14. One of them, b r o m o - b e n z o d i a z e p i n e (7-bromo-5-(2-fluorophenyl)methyl- 1 , 3 - d i h y d r o - 2 H - 1 , 4 - b e n z o d i a z e p i n - 2 - o n e ) ,
has now been labeled with tritium in the 1-methyl position, and its t r a n s p o r t and distribution in rat brain have been evaluated by regional autoradiographic techniques. B r o m o - b e n z o d i a z e p i n e ( B F B ) is rapidly taken up by brain in an a p p a r e n t l y irreversible m a n n e r during the time course of the experiments. T h e transport constant (clearance, K'in) is indistinguishable from the cerebral b l o o d flow rate, and the b r a i n - t o - b l o o d partition coefficient is i m m e a s u r e a b l y large. The distribution of BFB in brain sections appears to be proportional to b l o o d flow, and B F B , therefore, appears to be an excellent blood flow m a r k e r , similar to that of flunitrazepam 6 or d i a z e p a m 16. MATERIALS AND METHODS The blood-to-brain t r a n s p o r t of BFB was investigated using 14 healthy, adult rats (BD IX, 200-300 g). The animals were induced and briefly anesthetized with 3% halothane, and catheters were placed in both femoral arteries and in one femoral
Correspondence: L.R. Drewes, Department of Biochemistry, School of Medicine, University of Minnesota, Duluth, MN 55812, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
56 vein. Following intubation, the animals were immobilized with curare, mechanically ventilated, and maintained on 0.8% halothane. Isotopes in this study included [U-14C]sucrose from New England Nuclear (Boston, MA) and [3H]BFB (6.5 Ci/mmol), which was tritiated from [3H]CH3I, analogously to a previously described methylation procedure 1°. Tissue, blood, and plasma samples were digested in 1.5 ml of 50% tissue solubilizer (Protosol, NEN) in n-butanol (v/v). After bleaching with 0.5 ml of 30% H202, the mixtures were incubated overnight at 40 °C. The mixtures were then neutralized with 0.75 ml of 0.5 N HC1, and scintillation fluid was added. The radioactivity levels of the samples were determined, and the specific activities were calculated using appropriate quench correction. Blood gases and pH of arterial blood samples (200 ~1) were determined using a microblood gas/pH analyzer (Radiometer, Copenhagen). Blood-to-brain transport of [3H]BFB was determined by the integral method as previously described 1"4'6. Basically, the method involves determining the amount of tracer present in brain tissue at various times after an intravenous bolus injection of the radioactive substance and is based on the following relationship: T
M*(T)-M*vasc(T) = K*m I"
C*a(t)dt
0
in which M*(T) is the amount of substance present in brain at time T, M*.... (7) is the amount of substance trapped in the vascular space of the tissue sample, v K'in is the influx constant, and [
C*,d(t)dt is
the
in which the left equality is the apparent volume of distribution (ml/g), and M*vasc(T)/C*a(7)is the vascular volume (ml/g) of the tissue. This relationship can be plotted; the resultant slope is equal to K'in , the influx constant (ml/100 g/min), and the y-intercept is the vascular volume. Experiments were conducted by preparing the animals and allowing them to come to respiratory steady states as determined by arterial blood gas analysis and continuous arterial blood pressure recordings. The experiments were then initiated by rapid (1 s) intravenous injection of a mixture of the test compound (20/~Ci of [3H]BFB) and intravascular marker (5 #Ci of [14C]sucrose) in 0.4 ml of physiological saline. Animals were decapitated at various times from 10-120 s after injection, and the brains were rapidly removed. The parietal cortex was sampled for isotope analysis. The concentration-time integral for the duration of the experiment was determined mechanically by constant withdrawal of arterial blood in a syringe pump. The total volume of arterial blood withdrawn was less than 80 Atl. For autoradiography only, [3H]BFB (160 1~Ci or 230 #Ci) was injected intravenously. The animal was decapitated either after 60 s or after 240 s, and the brain was quickly removed. The entire brain was immediately immersed in methylbutane at -60 °C and transferred to a cryostat at -20 °C for preparation of 20-Arm thick sections. The sections were dried, placed in an X-ray cassette holder against tritium-sensitive film (Ultrafilm, LKB) and exposed for 21 days. The autoradiograms were then evaluated by digitizing the optical densities and displaying them as color-coded images using a computerized (PDP-11) and rotating densitometer (Scandig 3, Joyce Loebl Co.).
d f)
integrated concentration-time curve for the time T. By rearranging and dividing both sides of the equation by C*~ (7), the concentration of substance in arterial blood at time T, the following equation is obtained: T
M*(7) C*~ (T) - K*m
f C*a(t)dt
Mva~c(T)
c*~ (7) + c*~ (:r)
RESULTS
AND
DISCUSSION
The animals included in this study were in respiratory steady states and were consistently in the physiologically normal range (Table I). The cerebral vasculature was extremely permeable to BFB. This diazepine entered the parietal cortex of rat brain at an influx rate constant (K*ln) of 83 ml/100 g/min and at an apparent distribution volume that continued to increase for the duration of the ex-
57 TABLE I
g/rain, respectively 5. The p e r m e a b i l i t y of [U-14C]su -
Summary of physiological data
crose was consistently low and in the range previously reported. Sucrose was, therefore, used as a m a r k e r in the studies r e p o r t e d here to calculate the t r a p p e d vascular volume. Most substances cross the brain e n d o t h e l i u m because of their lipid solubility, a p r o p e r t y that may be expressed as the o c t a n o l : w a t e r partition coefficient (P) and m a y be d e t e r m i n e d empirically or estimated by calculation 7. Cerebrovascular p e r m e a b i l i t y is directly p r o p o r t i o n a l to log pS,13. The calculated log P values for B F B , d i a z e p a m , and flunitrazepam are in descending order, and B F B , therefore, has the greatest lipophilic character and would be expected to p e n e t r a t e the cerebrovasculature most easily. The a p p a r e n t distribution volume of BFB did not reach a m a x i m u m at the concentrations and times used in this study. The high p e r m e a b i l i t y suggests that BFB may be an excellent blood flow indicator. The result d e m o n s t r a t e d that delivery of B F B to brain tissue was a function only of b l o o d flow and was not diffusion-limited. A u t o r a d i o g r a p h y of sections from brain exposed to a bolus of [3H]BFB for 60 s indicated a distribution pattern similar to that of cerebral b l o o d flow (Fig. 2). There were no obvious areas of high concentrations or structures i m p e r m e a b l e to BFB. This indicated that BFB was distributed throughout the brain and that the areas known to contain high-affinity specific receptors for benzodiazepines 3 did not concentrate or preferentially trap the benzodiazepine derivative used at the time intervals and u n d e r the e x p e r i m e n t a l conditions of this study. The use of B F B or similar c o m p o u n d s as b l o o d flow tracers requires further validation. To use a benzodiazepine as a flow tracer under pathophysiological conditions would further require that the binding (trapping) of the agent be neither disturbed nor altered. BFB, during these experiments, e n t e r e d the tissue and presumably b o u n d to a combination of binding sites, either specific or non-specific. The specific receptors may include sites which potentially exhibit a pharmacological response (receptors) and sites which have no a p p a r e n t pharmacological activity (acceptors) 3. Consequently, the p a t t e r n of BFB distribution in brains used in these experiments cannot be interpreted to illustrate BFB receptors associated
Values are means +_S.E.M. in = 14) GLC~.rt(mM) pH pO e pCO 2 MBP~r~ Hct
10.9 _+ 1.0 mM* 7.41 + 0.02 108 + 8 mm Hg 43.2 -+ 1.2 mm Hg 93 _+5mmHg 40.7 _+0.5%
*n=ll.
perimental period investigated (Fig. 1). The K* m was about the same as o t h e r substances that are essentially completely flow-dependent, such as iodoantipyrine (83 ml/100 g/min), water (81 ml/100 g/min), and flunitrazepam (67 ml/100 g/min) 6. F o r comparison, the K*,,, for other less p e r m e a b l e substances such as sucrose (Fig. 1), mannitol, and D-glucose are 0.075 ml/100 g/rain, 0.25 ml/100 g/min, and 15 ml/100
10-
f
98.
7 ,.&
6
E
54. 32 1 0
;
i
i
;
8 lb
thota (rain)
Fig. 1. Graphical evaluation of blood-to-brain transfer constant. The apparent volume of distribution (ordinate) is plotted vs the arterial blood radioactivity integral/final plasma radioactivity (abscissa). The latter is designated theta and has the units min. The circles are the data points for BFB, and the line is the best fit by linear regression analysis. The slope is equivalent to the K*ln. The squares represent data from Gjedde et al. 6 for the BZD, flunitrazepam. The triangles are data points for blood-brain transport of sucrose which were determined simultaneously with the BFB data.
58
Fig. 2. Color pictorial images of [3H]BFB distribution in brain. Shown are coronal sections through the rat midbrain and cerebellum areas, The two images are on different scales with respect to size. Absorbance units x 100 are given on the color scale at the right,
with the G A B A receptors. Nevertheless, the ability to synthesize BFB labeled with the positron-emitting isotope 75BR (refs. 10,14) and the availability of specific benzodiazepine receptor antagonists make mapping-specific receptor populations in h u m a n subjects under normal and pathological conditions an attractive possibility.
ACKNOWLEDGEMENTS The authors are indebted to Dr. J. H a n u s for tritium labeling and to Dr. H. Scholl of providing the cold starting material. They also thank Carolyn Clark for editorial assistance in preparation of the manuscript. This research was supported in part by the Alexander yon H u m b o l d t F o u n d a t i o n , B o n n , Federal Republic of G e r m a n y .
REFERENCES 1 Blasberg, R.G., Fenstermacher, J.D. and Patlak, C.S., Transport of ct-aminoisobutyric acid across brain capillary and cellular membranes, J. Cerebr. Blood Flow Metabol., 3 (1983) 8-32. 2 Braestrup, C., Nielsen, M. and Honore, T., Binding of [3H]DMCM, a convulsive benzodiazepine ligand, to rat brain membranes: preliminary studies, J, Neurochem., 41 (1983) 454-465. 3 Gee, K.W., Ehlert, F.J., Roeske, W.R. and Yamamura, H.I., Heterogeneity of benzodiazepine receptors. In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 6, Plenum Press, New York, 1984, pp. 575-593. 4 Giedde, A., Rapid steady-state analysis of blood-brain glucose transfer in rat, Acta Physiol. Scand., 108 (1980) 331-339. 5 Gjedde, A., High- and low-affinity transport of D-glucose from blood to brain, J. Neurochem., 38 (1981) 1463-1471.
6 Gjedde, A., Drewes, L.R. and Christensen, B., 111.2. Brain uptake of a 'fluid microsphere': comparison of flunitrazepam, water, and iodoantipyrine transfer across the blood-brain barrier, J. Cerebr. Blood Flow Metabol., 3 (1983) 8-32. 7 Leo, A., Hansch, C. and Elkins, D., Partition coefficients and their uses, Chem. Rev., 71 (1971) 525-616. 8 Levin, V.A,, Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability, J. Med. Chem., 23 (1980)682-684. 9 Maziere, M., Godot, J.-M., Berger, G., Baron, J.C., Comar, D., Cepeda, C., Menini, C. and Naquet, R., Positron tomography, A new method for in vivo brain studies of benzodiazepine in animal and in man, Adv. Biochem. Psychopharmacol., 26 (1981) 273-286. 10 Maziere, M., Godot, J.-M., Berger, G., Prenant, C.H. and Comar, D., High specific activity C-11 labeling of benzodiazepines diazepam and flunitrazepam, J. Radioanal. Chem,, 56 (1980) 229-235.
59 11 Mohler, H. and Okada, T., Properties of diazepam-H-3 binding to benzodiazepine receptors in rat cerebral-cortex, Life Sci., 20 (1977) 2101-2110. 12 Nielsen, M., Schou, H. and Braestrup, C., [3H]Propyl-/3carboline-3-earboxylate binds specifically to brain benzodiazepines receptors, J. Neurochem., 36 (1981) 276-285. 13 Rapoport, S.I., Ohno, K. and Pettigrew, K.D., Drug entry in the brain, Brain Research, 172 (1979) 354-359. 14 Scholl, H., Kloster, G. and Stfcklin, G., Bromine-75labeled 1,4-benzodiazepines: potential agents for the map-
ping of benzodiazepine receptors in vivo: concise communication, J. Nucl. Med., 24 (1983) 417-422. 15 Squires, R.F. and Braestrup, C.. Benzodiazepine receptors in rat brain, Nature (London), 266 (1977) 732-734. 16 Takasato, Y., Rapoport, S.I. and Smith, Q.R., An in situ brain perfusion technique to study cerebrovascular transport in the rat, Am. J. Physiol., 247 (1984) H484-H493. 17 Ticku, M.K. and Macksay, G., Convulsant/depressant site of action at the allosteric benzodiazepine-GABA receptorionophore complex, Life Sci., 33 (1983) 2363-2375.
55
BRE 12263
Blood-brain transport and regional distribution of bromo-benzodiazepine Lester R. D r e w e s l, G u n t e r Mies 2, K.-A. H o s s m a n n 2 and G e r h a r d St6cklin 3 1Department of Biochemistry, School of Medicine, University of Minnesota, Duluth, MN55812 (U.S.A.), 2Max-Planck Institute for Brain Research, Cologne and 3Institute of Chemistry, KFA Jiilich, Jiilich (F.R. G.) (Accepted 27 May 1986)
Key words: Benzodiazepine; Transport; Blood-Brain; Receptor; Blood flow; Autoradiography; Integral method
The blood-brain transport and regional distribution of a tritium-labeled, brominated benzodiazepine (BFB) was determined for the rat brain in vivo. The unidirectional transport constant from blood to brain was measured by a graphical, integral method and was found to be 0.83 ml/g/min, a value which indicates that transport is essentially flow-dependent. The apparent volume of distribution increased linearly during the measurement period, suggesting that back transport from brain to blood was zero and that BFB was trapped in the tissue, possibly by specific receptors or acceptors. Under the conditions of these experiments, autoradiography of brain tissue sections indicated a regional distribution of [3H]BFB similar to that expected for regional cerebral blood flow. These results indicate that BFB is a useful blood flow tracer in brain and suggest that BFB radiobrominated with bromine-75 may, under appropriate conditions, be a suitable tracer for in vivo regional blood flow measurement or benzodiazepine receptor mapping by positron-emission tomography.
INTRODUCTION During the past two decades benzodiazepine ( B Z D ) drugs such as d i a z e p a m have gained remarkable popularity as anxiolytics, sedative anticonvulsants, and muscle relaxants. In recent years their mechanisms of action have b e e n investigated, and recent studies have focused on specific high-affinity binding sites for these drugs in the m a m m a l i a n nervous system 11'a5. These binding sites now a p p e a r to be linked in some yet undefined way to the postsynaptic G A B A r e c e p t o r and associated chloride ionophore 17. In vitro binding studies have shown regional differences in the distribution of B Z D binding sites. Cerebral cortex has about 2.5 times the r e c e p t o r density as the cerebellum 2'12. H o w e v e r , few in vivo studies have been r e p o r t e d 9. Recently, 1,4-benzodiazepines were labeled with the isotope [75Br] as potential agents for mapping receptors in vivo 14. One of them, b r o m o - b e n z o d i a z e p i n e (7-bromo-5-(2-fluorophenyl)methyl- 1 , 3 - d i h y d r o - 2 H - 1 , 4 - b e n z o d i a z e p i n - 2 - o n e ) ,
has now been labeled with tritium in the 1-methyl position, and its t r a n s p o r t and distribution in rat brain have been evaluated by regional autoradiographic techniques. B r o m o - b e n z o d i a z e p i n e ( B F B ) is rapidly taken up by brain in an a p p a r e n t l y irreversible m a n n e r during the time course of the experiments. T h e transport constant (clearance, K'in) is indistinguishable from the cerebral b l o o d flow rate, and the b r a i n - t o - b l o o d partition coefficient is i m m e a s u r e a b l y large. The distribution of BFB in brain sections appears to be proportional to b l o o d flow, and B F B , therefore, appears to be an excellent blood flow m a r k e r , similar to that of flunitrazepam 6 or d i a z e p a m 16. MATERIALS AND METHODS The blood-to-brain t r a n s p o r t of BFB was investigated using 14 healthy, adult rats (BD IX, 200-300 g). The animals were induced and briefly anesthetized with 3% halothane, and catheters were placed in both femoral arteries and in one femoral
Correspondence: L.R. Drewes, Department of Biochemistry, School of Medicine, University of Minnesota, Duluth, MN 55812, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
56 vein. Following intubation, the animals were immobilized with curare, mechanically ventilated, and maintained on 0.8% halothane. Isotopes in this study included [U-14C]sucrose from New England Nuclear (Boston, MA) and [3H]BFB (6.5 Ci/mmol), which was tritiated from [3H]CH3I, analogously to a previously described methylation procedure 1°. Tissue, blood, and plasma samples were digested in 1.5 ml of 50% tissue solubilizer (Protosol, NEN) in n-butanol (v/v). After bleaching with 0.5 ml of 30% H202, the mixtures were incubated overnight at 40 °C. The mixtures were then neutralized with 0.75 ml of 0.5 N HC1, and scintillation fluid was added. The radioactivity levels of the samples were determined, and the specific activities were calculated using appropriate quench correction. Blood gases and pH of arterial blood samples (200 ~1) were determined using a microblood gas/pH analyzer (Radiometer, Copenhagen). Blood-to-brain transport of [3H]BFB was determined by the integral method as previously described 1"4'6. Basically, the method involves determining the amount of tracer present in brain tissue at various times after an intravenous bolus injection of the radioactive substance and is based on the following relationship: T
M*(T)-M*vasc(T) = K*m I"
C*a(t)dt
0
in which M*(T) is the amount of substance present in brain at time T, M*.... (7) is the amount of substance trapped in the vascular space of the tissue sample, v K'in is the influx constant, and [
C*,d(t)dt is
the
in which the left equality is the apparent volume of distribution (ml/g), and M*vasc(T)/C*a(7)is the vascular volume (ml/g) of the tissue. This relationship can be plotted; the resultant slope is equal to K'in , the influx constant (ml/100 g/min), and the y-intercept is the vascular volume. Experiments were conducted by preparing the animals and allowing them to come to respiratory steady states as determined by arterial blood gas analysis and continuous arterial blood pressure recordings. The experiments were then initiated by rapid (1 s) intravenous injection of a mixture of the test compound (20/~Ci of [3H]BFB) and intravascular marker (5 #Ci of [14C]sucrose) in 0.4 ml of physiological saline. Animals were decapitated at various times from 10-120 s after injection, and the brains were rapidly removed. The parietal cortex was sampled for isotope analysis. The concentration-time integral for the duration of the experiment was determined mechanically by constant withdrawal of arterial blood in a syringe pump. The total volume of arterial blood withdrawn was less than 80 Atl. For autoradiography only, [3H]BFB (160 1~Ci or 230 #Ci) was injected intravenously. The animal was decapitated either after 60 s or after 240 s, and the brain was quickly removed. The entire brain was immediately immersed in methylbutane at -60 °C and transferred to a cryostat at -20 °C for preparation of 20-Arm thick sections. The sections were dried, placed in an X-ray cassette holder against tritium-sensitive film (Ultrafilm, LKB) and exposed for 21 days. The autoradiograms were then evaluated by digitizing the optical densities and displaying them as color-coded images using a computerized (PDP-11) and rotating densitometer (Scandig 3, Joyce Loebl Co.).
d f)
integrated concentration-time curve for the time T. By rearranging and dividing both sides of the equation by C*~ (7), the concentration of substance in arterial blood at time T, the following equation is obtained: T
M*(7) C*~ (T) - K*m
f C*a(t)dt
Mva~c(T)
c*~ (7) + c*~ (:r)
RESULTS
AND
DISCUSSION
The animals included in this study were in respiratory steady states and were consistently in the physiologically normal range (Table I). The cerebral vasculature was extremely permeable to BFB. This diazepine entered the parietal cortex of rat brain at an influx rate constant (K*ln) of 83 ml/100 g/min and at an apparent distribution volume that continued to increase for the duration of the ex-
57 TABLE I
g/rain, respectively 5. The p e r m e a b i l i t y of [U-14C]su -
Summary of physiological data
crose was consistently low and in the range previously reported. Sucrose was, therefore, used as a m a r k e r in the studies r e p o r t e d here to calculate the t r a p p e d vascular volume. Most substances cross the brain e n d o t h e l i u m because of their lipid solubility, a p r o p e r t y that may be expressed as the o c t a n o l : w a t e r partition coefficient (P) and m a y be d e t e r m i n e d empirically or estimated by calculation 7. Cerebrovascular p e r m e a b i l i t y is directly p r o p o r t i o n a l to log pS,13. The calculated log P values for B F B , d i a z e p a m , and flunitrazepam are in descending order, and B F B , therefore, has the greatest lipophilic character and would be expected to p e n e t r a t e the cerebrovasculature most easily. The a p p a r e n t distribution volume of BFB did not reach a m a x i m u m at the concentrations and times used in this study. The high p e r m e a b i l i t y suggests that BFB may be an excellent blood flow indicator. The result d e m o n s t r a t e d that delivery of B F B to brain tissue was a function only of b l o o d flow and was not diffusion-limited. A u t o r a d i o g r a p h y of sections from brain exposed to a bolus of [3H]BFB for 60 s indicated a distribution pattern similar to that of cerebral b l o o d flow (Fig. 2). There were no obvious areas of high concentrations or structures i m p e r m e a b l e to BFB. This indicated that BFB was distributed throughout the brain and that the areas known to contain high-affinity specific receptors for benzodiazepines 3 did not concentrate or preferentially trap the benzodiazepine derivative used at the time intervals and u n d e r the e x p e r i m e n t a l conditions of this study. The use of B F B or similar c o m p o u n d s as b l o o d flow tracers requires further validation. To use a benzodiazepine as a flow tracer under pathophysiological conditions would further require that the binding (trapping) of the agent be neither disturbed nor altered. BFB, during these experiments, e n t e r e d the tissue and presumably b o u n d to a combination of binding sites, either specific or non-specific. The specific receptors may include sites which potentially exhibit a pharmacological response (receptors) and sites which have no a p p a r e n t pharmacological activity (acceptors) 3. Consequently, the p a t t e r n of BFB distribution in brains used in these experiments cannot be interpreted to illustrate BFB receptors associated
Values are means +_S.E.M. in = 14) GLC~.rt(mM) pH pO e pCO 2 MBP~r~ Hct
10.9 _+ 1.0 mM* 7.41 + 0.02 108 + 8 mm Hg 43.2 -+ 1.2 mm Hg 93 _+5mmHg 40.7 _+0.5%
*n=ll.
perimental period investigated (Fig. 1). The K* m was about the same as o t h e r substances that are essentially completely flow-dependent, such as iodoantipyrine (83 ml/100 g/min), water (81 ml/100 g/min), and flunitrazepam (67 ml/100 g/min) 6. F o r comparison, the K*,,, for other less p e r m e a b l e substances such as sucrose (Fig. 1), mannitol, and D-glucose are 0.075 ml/100 g/rain, 0.25 ml/100 g/min, and 15 ml/100
10-
f
98.
7 ,.&
6
E
54. 32 1 0
;
i
i
;
8 lb
thota (rain)
Fig. 1. Graphical evaluation of blood-to-brain transfer constant. The apparent volume of distribution (ordinate) is plotted vs the arterial blood radioactivity integral/final plasma radioactivity (abscissa). The latter is designated theta and has the units min. The circles are the data points for BFB, and the line is the best fit by linear regression analysis. The slope is equivalent to the K*ln. The squares represent data from Gjedde et al. 6 for the BZD, flunitrazepam. The triangles are data points for blood-brain transport of sucrose which were determined simultaneously with the BFB data.
58
Fig. 2. Color pictorial images of [3H]BFB distribution in brain. Shown are coronal sections through the rat midbrain and cerebellum areas, The two images are on different scales with respect to size. Absorbance units x 100 are given on the color scale at the right,
with the G A B A receptors. Nevertheless, the ability to synthesize BFB labeled with the positron-emitting isotope 75BR (refs. 10,14) and the availability of specific benzodiazepine receptor antagonists make mapping-specific receptor populations in h u m a n subjects under normal and pathological conditions an attractive possibility.
ACKNOWLEDGEMENTS The authors are indebted to Dr. J. H a n u s for tritium labeling and to Dr. H. Scholl of providing the cold starting material. They also thank Carolyn Clark for editorial assistance in preparation of the manuscript. This research was supported in part by the Alexander yon H u m b o l d t F o u n d a t i o n , B o n n , Federal Republic of G e r m a n y .
REFERENCES 1 Blasberg, R.G., Fenstermacher, J.D. and Patlak, C.S., Transport of ct-aminoisobutyric acid across brain capillary and cellular membranes, J. Cerebr. Blood Flow Metabol., 3 (1983) 8-32. 2 Braestrup, C., Nielsen, M. and Honore, T., Binding of [3H]DMCM, a convulsive benzodiazepine ligand, to rat brain membranes: preliminary studies, J, Neurochem., 41 (1983) 454-465. 3 Gee, K.W., Ehlert, F.J., Roeske, W.R. and Yamamura, H.I., Heterogeneity of benzodiazepine receptors. In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 6, Plenum Press, New York, 1984, pp. 575-593. 4 Giedde, A., Rapid steady-state analysis of blood-brain glucose transfer in rat, Acta Physiol. Scand., 108 (1980) 331-339. 5 Gjedde, A., High- and low-affinity transport of D-glucose from blood to brain, J. Neurochem., 38 (1981) 1463-1471.
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